Understanding the structural basis of replication initiation in AAV

了解 AAV 复制起始的结构基础

• 批准号: 8349753
• 负责人: Frederick Dyda
• 金额: $ 35.23万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8349753
• 关键词: Active Sites; Amyotrophic Lateral Sclerosis; Binding; Binding Sites; Bovine papillomavirus E1 protein; Cells; Characteristics; Childhood; Chromosomes, Human, Pair 19; Cleaved cell; Complex; Crystallization; Cystic Fibrosis; DNA; DNA Integration; DNA Sequence; DNA biosynthesis; DNA-dependent ATPase-endonuclease; Dependovirus; Diabetes Mellitus; Disease; Disease remission; Electron Microscopy; Enzymes; Exhibits; Foundations; Gene Transduction Agent; Genes; Genome; Goals; Hemophilia A; Human; Human Chromosomes; Insulin; Ions; Lead; Light; Location; Mediating; Medical; Membrane Proteins; Metals; Modeling; Molecular; Mus; Nature; Nucleotides; Oligonucleotides; Parvovirus; Process; Property; Proteins; Rattus; Replication Initiation; Replication-Associated Process; Reporting; Research; Resolution; SV40 T Antigens; Shorthand; Sickle Cell Anemia; Single-Stranded DNA; Site; Specificity; Structure; Surface; System; Tetranucleotide Repeat; Time; Tyrosine; Viral; Viral Genome; Viral Proteins; Virus; analog; base; design; endonuclease; gene correction; gene therapy; helicase; human DNA; human disease; improved; interest; member; nuclease; prevent; programs; site-specific integration; structural biology; success; vector; viral DNA

The structure of the Rep endonuclease domain (Hickman et al., 2002) revealed that it is unrelated to all other structurally characterized nucleases and provided the first view of an HUH superfamily member. Rep is homologous to the origin binding domains of the SV40 T antigen (Luo et al., 1996) and replication initiation protein E1 of bovine papillomavirus (Enemark et al., 2000). The HUH residues, which bind the catalytically required metal ion, converge with a helix bearing the two active site tyrosine residues to create the enzyme active site cleft. Subsequent co-crystal structures of the Rep nuclease domain with oligonucleotides representing two specific regions of the AAV genome showed that the nuclease domain uses two different protein surfaces to recognize its DNA target. One surface binds a hairpin that is at the very tip of the viral genome and the other recognizes a repeated tetranucleotide sequence close to the genome ends that constitutes the Rep binding site. These structures allowed us to propose a model for the assembly of a hexameric Rep-DNA complex that is poised to nick the viral DNA and begin unwinding it as a prelude to replication. It seems likely that site-specific integration also begins with a nick at a related sequence in human chromosome 19. Although it is believed that Rep assembles as a hexameric helicase, such assemblies had not previously been observed and the mode of Rep multimerization remains controversal. We have recently been studying an N-terminally truncated version of Rep in which the endonuclease domain is missing. This portion of Rep assembles as a hexamer on both single-stranded and dsDNA substrates, and is not dependent on specific viral DNA sequences or on the presence of nucleotides. We have been able to directly visualize these hexameric complexes using electron microscopy, and crystallization trials are underway. Enemark, E.J., Chen, G., Vaughn, D.E., Stenlund, A., and Joshua-Tor, L. (2000) Mol. Cell 6, 149-158. Flotte, T.R. (2005) Pediatric Res. 58, 1143-1147. Hickman, A.B., Ronning, D.R., Kotin, R.M., and Dyda, F. (2002) Mol. Cell 10, 327-337. Im, D.S. and Muzyczka, N. (1990) Cell 61, 447-457. Le Bec, C. and Douar, A.M. (2006) Gene Ther. 13, 805-813. Lee, H.C., Kim, S.J., Kim, K.S., Shin, H.CV., and Yoon, J. W. (2000) Nature 408, 483-488. Luo, X., Sanford, D.G., Bullock, P.A., and Bachovchin, W.W. (1996) Nat. Struct. Biol. 3, 1034-1039.

Rep核酸内切酶结构域的结构（Hickman等人，2002）揭示了它与所有其他结构特征的核酸酶无关，并提供了HUH超家族成员的第一个观点。Rep与SV 40 T抗原的起始结合结构域同源（Luo等人，1996）和牛乳头瘤病毒的复制起始蛋白E1（Enemark等，2000年）。结合催化所需的金属离子的HUH残基与带有两个活性位点酪氨酸残基的螺旋会聚以产生酶活性位点裂缝。随后Rep核酸酶结构域与代表AAV基因组的两个特定区域的寡核苷酸的共晶体结构显示核酸酶结构域使用两个不同的蛋白质表面来识别其DNA靶标。一个表面结合位于病毒基因组最顶端的发夹，另一个表面识别靠近基因组末端的重复四核苷酸序列，该序列构成Rep结合位点。这些结构使我们能够提出一个组装六聚体Rep-DNA复合物的模型，该复合物准备切割病毒DNA并开始解旋，作为复制的前奏。位点特异性整合似乎也始于人类19号染色体相关序列的切口。 尽管据信Rep组装为六聚体解旋酶，但以前未观察到这样的组装，并且Rep多聚化的模式仍然存在争议。我们最近一直在研究一个N-末端截短版本的Rep中的核酸内切酶结构域是失踪。Rep的这一部分在单链和dsDNA底物上组装为六聚体，并且不依赖于特定的病毒DNA序列或核苷酸的存在。 我们已经能够使用电子显微镜直接观察这些六聚体复合物，结晶试验正在进行中。 Enemark，E.J.，陈，G.，Vaughn，D.E.，Stenlund，A.，和Joshua-Tor，L.（2000）Mol. 6号牢房149-158 Flotte，T.R.等（2005）Pediatric Res. 58，1143-1147。 Hickman，A. B.，Ronning，D.R.，Kotin，R.M.，和Dyda，F.等人（2002）Mol. 10号牢房327-337 我，D.S.和Muzyczka，N.等（1990）Cell 61，447-457. 勒贝克角和Douar，A.M.（2006）Gene Ther. 13，805-813。 Lee，H.C.，Kim，S.J.，金，K.S.，Shin，H.C.，和Yoon，J.W.等人（2000）Nature 408，483-488. Luo，X.，桑福德，D.G.，布洛克，私人助理，和Bachovchin，W. W.等（1996）Nat.Struct.Biol.3，1034-1039。